Self-contained devices for detecting biological contaminants

ABSTRACT

The present invention relates generally to self-contained devices and methods for detecting biological contaminants and, more particularly, to self-contained devices and methods for detecting biological contaminants in relevant settings including, for example, food processing plants, hospitals, medical offices, veterinary offices, and restaurants by using a device or methodology that includes a sampler component and dye component that binds to the biological material in a detectable fashion.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S ProvisionalApplication No. 60/208,865, filed Jun. 2, 2000.

TECHNICAL FIELD

[0002] The present invention relates generally to self-contained devicesand methods for detecting biological contaminants and, moreparticularly, to self-contained devices and methods for detectingbiological contaminants in relevant settings including food processingplants, hospitals, medical offices, veterinary offices, and restaurantsby using a device or methodology that includes a sampler component anddye component that binds to the biological material in a detectablefashion.

BACKGROUND OF THE INVENTION

[0003] This invention relates to the field of testing for biologicalcontaminants. Such contaminants may be found on equipment or othersurfaces used in environments including food processing plants,hospitals, veterinary offices, and restaurants when a thorough cleaninghas not been performed. Materials detected as indicators ofcontamination include, for example, viable bacterial cells, viruses,ATP, and protein. The ideal test for contamination would be effective,inexpensive, rapid, and easy to use and interpret.

[0004] Although pathogenic bacteria and viruses are clearly hazardous,the commonly used methods to detect them involve a number of steps, suchas reagent preparation, reagent mixing, sample transfer, sample/reagentmixing, and incubation prior to reading results. Tests for thesematerials are not only time consuming but they also typically requirethe use of expensive and complicated equipment. Thus, routine testing ofsurfaces or equipment directly for bacteria or viruses is not practicalin settings in which the testing is done by non-technical personnel andrapid results are demanded. As an alternative to testing for bacteriaand viruses, many food processing plants use surrogate markers such asATP and protein as indicators of contamination. Although thesesubstances themselves are not usually harmful, they may serve asnutrient material for bacteria and are good general indicators of theeffectiveness of cleaning.

[0005] ATP (adenosine triphosphate) is a chemical common to all livingorganisms. The presence of significant levels of ATP on a surfaceindicates that cleaning was incomplete and that bacteria may be present.Because hygiene monitoring by ATP detection is common, relativelyinexpensive, rapid, and effective, it is widely used. However, detectingATP suffers from certain limitations that restrict its utility, in thatit involves the use of a relatively expensive instrument and may involvetransporting samples to a central laboratory.

[0006] Clearly tests for protein as evidence of contamination provide anattractive alternative to direct testing for bacteria and viruses or forATP detection. Unfortunately, many examples of current methods forprotein determination, which can serve as an indicator of a contaminatedsurface involve on-site reagent preparation due to stability problems,along with multiple transfer steps, incubation periods, and/or involvehighly subjective color changes which make interpretation difficult,and/or require the use of complex instrumentation or specially trainedpersonnel. Examples of common protein determination methods aredescribed in Stoscheck, Quantitation of Protein, in METHODS INENZYMOLOGY Vol. 182, pp.50-68, 1990. Among the variants of basic proteindetection methods are methods using colloidal forms of COOMASSIE® bluestain to detect proteins in gels such as polyacrylamide electrophoresisgels. Such methods are described, for example, in Neuhoff et al.,Electrophoresis 6:427-488, 1985 and Neuhoff et al., Electrophorests9:255-262, 1988. In addition to the conventional protein assay methodsreferenced above, a combination cleaning and protein stainingcomposition is described in Winicov et al., U.S. Pat. No. 5,424,000,entitled ACID CLEANINGS AND STAINING COMPOSITIONS, issued Jun. 13, 1995.The solutions preferably include phosphoric, sulfuric, and nitric acids,and Acid Violet 19 dye.

[0007] A number of different self-contained sampling/testing devicesemploying certain assays have been described. Examples of such assaysinclude sampling for bacterial contaminants in food processing plants,the sampling for contamination of the environment by heavy metals suchas lead, and the collection of specimens from a patient to test formicroorganism infection.

[0008] Specific examples of self-contained sampling/testing devicesinclude Nason, U.S. Pat. No. 5,266,266, issued Nov. 30, 1993, and Nason,U.S. Pat. No. 4,978,504, issued Dec. 18, 1990, both entitled SPECIMENTEST UNIT; Nason, U.S. Pat. No. 4,707,450, issued Nov. 17, 1987,entitled SPECIMEN COLLECTION AND TEST UNIT; Numa, U.S. Pat. No.5,726,062, issued Mar. 10, 1998; and Tobin, U.S. Pat. No. 3,792,699,issued Feb. 19, 1974. The use of protein error dyes to estimate proteincontent in urine is disclosed in Keston, U.S. Pat. No. 3,485,587,entitled PROTEIN INDICATOR. All of which are hereby incorporated byreference in their entireties including drawings. However, all of theaforementioned devices suffer from the same drawbacks noted above.

[0009] Thus, as no self-contained device or methodology exists that isinexpensive, rapid, and easy to use and interpret, there exists a needin the art for such a device relevant to the detection of biologicalcontamination. The present invention fulfills these needs, and providesother related advantages.

SUMMARY OF THE INVENTION

[0010] The present invention generally provides novel methods andself-contained devices for detecting biological contaminants in avariety of settings. In one aspect, provided is a self-containedsampling/testing device having a sampler for collecting target materialand a signal generator comprising a dye which binds to said targetmaterial to signal the presence of said target material. In oneembodiment the device also contains a sampler washer having a washsolution. In other embodiments, the device has an absorbent material,wherein the sampler has a porous sample collection pad, and theabsorbent material, the sampler, and the sampler washer are configuredand arranged such that the sample collection pad can be disposed betweenthe absorbent material and the sampler washer so that the wash solutionseparates dye bound to said target material immobilized on said samplecollection pad from unbound dye.

[0011] Also included are embodiments wherein the sampler has a poroussample collection pad for collecting the target material, wherein thedye and the wash solution are contained in a reagent tray housingcapable of being contacted by the sampler to impart the dye and the washsolution across the target material contained on the sampler. Furtherembodiments include a device wherein the dye and the wash solution arecontained in a plurality of reservoirs, wherein the reservoirs areserially contacted by the sampler to first expose the target material tothe dye and then to wash unbound dye away from bound dye. In relatedembodiments the device may contain a wetting agent for moistening saidsample collection pad in advance of collecting said target material.Further, the wetting agent may be the same as or differ from the washsolution.

[0012] In yet other embodiments the device has a sampler that is hollowand wherein the absorbent material is disposed within the sampler tofacilitate transport of the dye and the wash solution across the targetmaterial or the wash solution is disposed within the sampler.

[0013] In one embodiment the device comprises a reagent housingcomprising an absorbent material, wherein one of the absorbent materialsis saturated with the wash solution, and the other of the absorbentmaterials is unsaturated, wherein unbound dye from a sampler collectionsurface disposed there between is washed by the flow of the washsolution from the saturated absorbent material to unsaturated absorbentmaterial. In addition, the dye may be transported by the wash solutionto the target material to effect the binding. Further, the device maycontain at least one rupturable membrane. This membrane may separate anycomponent such as wetting solution from the absorbent material, dye fromthe absorbent material, or wash solution from the absorbent material. Incertain embodiments the dyes utilized may be protein binding dyes suchas Ponceau-S and in other embodiments dyes which either precipitate andstain target material or change color (frequency shift) upon bindingtarget material.

[0014] In other embodiments the device contains a dye in a dry formuntil contacted by a wetting agent or sample. In still other embodimentsthe device may contain a neutralizing agent to neutralize any compoundsin the sample that might interfere with the binding of the dye to thetarget material. In certain embodiments the neutralizing agent is sodiumthiosulfate, MgCl₂, sodium dodecyl sulfate, tergitol, Triton X-100, orTween 20. In yet other embodiments the dye is a frequency shift dye orcolloidal dye. In specific embodiments these dyes may be Coomassie dyesor bromophenol blue.

[0015] In the various embodiments, the sampler is contained within alower housing providing protection from pre-testing contamination forthe sampler, the device further comprising an upper housing, wherein theupper housing and the lower housing sealably engage, and the sampler isattached to the upper housing. In other embodiments the sampler of thedevice contains an absorbent pad at the surface of which is positioned amembrane to which a dye is attached either covalently or non covalentlyor in the alternative the dye is attached directly to the absorbent padand/or the no membrane is present.

[0016] In other aspects the device contains a wetting/neutralizingsolution that is contained within a reagent housing containing anabsorbent material. In further embodiments the sampler contains anabsorbent material which is pre moistened with wetting/neutralizingsolution. In the alternative the sampler contains a breakable vial orrupturable compartment containing wetting/neutralizing solution.Further, the device of the invention may contain a frequency shift dyeof the protein error family, such as bromophenol blue. In furtherembodiments, the wetting/neutralizing solution may be sodiumthiosulfate, MgCl₂, sodium dodecyl sulfate, tergitol, Triton X- 100, orTween 20.

[0017] In a further aspect, a self-contained sampling/testing device isprovided which comprises a sampler for collecting biological targetmaterial and a signal generator comprising a frequency shift dye of theprotein error family, the dye being capable of binding to the biologicaltarget material and producing a color change upon binding to the targetmaterial wherein unbound frequency shift dye need not be separated frombiological target bound frequency shift dye in order to detect thepresence of target material. In related embodiments the device containsat least one of the following: an absorbent material and/or a wettingagent. In certain embodiments the dye is bromophenol blue, while inother embodiments the device contains a wetting solution that contains aneutralizing agent such as one or more of sodium thiosulfate, MgCl₂,sodium dodecyl sulfate, tergitol, Triton X-100, or Tween 20.

[0018] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andexamples. In addition, the various references set forth below describein more detail certain procedures or compositions (e.g., dyes, detectionmethodologies, etc.), and are therefore each incorporated herein, byreference, in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 illustrates an embodiment in the published art containing asampler wand with an absorbent sample collection pad, and a foldablebook encompassing reservoir pads for dye, absorbent and wash solution.

[0020]FIG. 2 illustrates an embodiment in the published art containing asampler wand with a sample collection pad and an absorbent backing, anda reagent dish with three wells: one with swab wetting solution, onewith dye, and one with washing solution.

[0021]FIG. 3 illustrates an embodiment in the published art containing asampler stick and a reagent dish with three wells; one with samplerwetting solution, one with dye, and one with wash solution.

[0022]FIG. 4 illustrates another embodiment to that of FIG. 3 where thereagent tray is simplified to two wells with the dye and wash reagentspresent in the same well separated by an impermeable but rupturablemembrane.

[0023]FIG. 5 illustrates an embodiment of a device in the published artwhere the wash solution is contained in a sampler stick separated fromthe sampling surface by an impermeable membrane. Prior to use, thesample collection end of the sampler stick sealably engages with acavity in a housing. The dye is present in the same cavity and separatedfrom the wash surface by a permeable membrane. The opposite end of thehousing contains an absorbent material and can be sealably engaged withthe collection end of the sampler stick following sample collection.

[0024]FIG. 6 illustrates an embodiment in the published art containing aswab-type sampler in which the dye solution in the sampler portion alsofunctions as the sampler wash solution.

[0025]FIG. 7 illustrates an embodiment in the published art of a devicein which the housing is divided into an upper housing portion and alower housing portion which sealably engage, in which the lower portionof the lower housing is set off by a separator.

[0026]FIG. 8 shows an embodiment in the published art of a device inwhich the upper portion of device contains a sampler and a sampler washsolution which contains no dye. The lower portion of the device (thehousing), protects the sampler when the two portions of the device aresealed together. The housing also contains a separator which divides thehousing into upper and lower spaces. The lower space contains a dyecomposition, preferably a dry dye.

[0027]FIG. 9 illustrates an embodiment of the inventive device in whichthe dye is bound to a membrane on the surface of the sampler. Thewetting solution is contained in a separate reservoir.

[0028]FIG. 10 illustrates an embodiment of the inventive device in whichthe dye is also bound to the surface of the membrane and the wettingagent is contained in a compartment separating it from the absorbentmaterial/dye-membrane by a breakable seal.

[0029]FIG. 11 illustrates an embodiment of the inventive device in whichthe dye is bound to a membrane at the surface of the sampler and thewetting agent is stored in a rupturable ampoule within the plastichousing of the sampler stick.

[0030]FIG. 12 illustrates an embodiment of the inventive device in whichthe dye is bound to a membrane at the surface of the sampler and thewetting agent is contained in the absorbent material within the samplerstick and each device is sealed in a foil pouch to prevent evaporationof the wetting agent.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The device and methods of the present invention areself-contained, inexpensive, rapid, and easy to use and interpret. Incertain embodiments, the device and methods utilize a dye that changescolor in the presence of significant amounts of protein (i.e., a proteinerror dye or polychromatic dye, see Keston et al., Supra) attached at ornear the surface of a sampling stick and in further embodiments thedevice or method contains a means for wetting the sampling surface priorto contact with the test surface. Such means for wetting can be providedby a wetting agent/solution which aids in protein solubilization,neutralizes contaminants that might interfere with protein detection,and maximizes detection of target material.

[0032] The need for, and utility of on site, immediate feedback tocleaning and audit personnel on the presence of residual contaminatingsubstances in a variety of environments is well-established. Forexample, the need for contaminant monitoring has a well-documented rolein food safety programs when residual food residues can result inbacterial contamination and allergic responses in some individuals.Effective cleaning also reduces the risk of pathogens contaminatingsubsequent food products. A variety of devices and methods have beenutilized for contaminant testing. Similarly, there is a need to ensurethat surfaces and equipment in hospitals, physicians' offices, clinicallaboratories, or veterinarian offices have been adequately cleaned toprotect patients and staff.

[0033] Particularly advantageous devices for the purpose of evaluatingthe presence of specific materials require no secondary reagents orsteps, have easily detected changes in the presence of target material,give immediate results, and allow integrated collection of sample intothe device. The present invention demonstrates that such aself-contained sampling/testing device can be constructed in which thepresence of target material in a sample is detected colorimetricallythrough use of a dye which binds the target material. As indicatedabove, this device is particularly advantageous for routine sanitationtesting procedures.

[0034] In a first aspect, the present invention concerns aself-contained device having a sampler for collecting a sample which maycontain a target material and a signal generator having a contactabledye that binds to the collected target material. In certain embodiments,the device may contain a sampler washer having a wash solution forwashing the collected target material and/or free dye from or on thesampler to facilitate measurement of a signal produced from theinteraction of dye and target material. In other related embodiments thedevice or method has a reservoir capable of providing a wetting agent tothe sampler. The sample collection surface portion of the sampler is incommunication with or can be placed in communication with the samplerwasher or sampler wetting agent. In certain embodiments, the samplercollection surface is also in communication with or can be placed incommunication with an absorbent material able to take up liquid from awetting agent and/or dye solution and/or wash solution. The device maybe constructed with any of many possible structural configurations,depending on the requirements of the particular application, e.g.,depending on the specific type of dye used and the type of targetmaterial to be tested.

[0035] The term “in communication,” as used herein, refers to a contactor channel or other means that allows fluid contact between thereferenced components. Thus, for example, a sampler washer and anabsorbent material are in communication if fluid transport can occurfrom the sampler washer or wetting agent reservoir into the absorbentmaterial. The term does not imply that fluid is actually present, butonly that such fluid contact could occur if fluid were present.

[0036] In certain embodiments, the device incorporates a target materialprecipitating dye, preferably a protein precipitating dye, for example,Ponceau-S dye. Such a dye binds to and precipitates, or assists inprecipitating or keeping out of solution a target material. The samplecollection surface of the sampler can be contacted with the dye (insolution or dry) in a manner such that a quantity sufficient to dyetarget material in a sample is taken up by the sampler. In using suchdyes, it is generally advantageous but not required to separate bounddye from unbound dye to provide convenient detection of the presence oftarget material. Thus, embodiments using such dyes may employ anarrangement where the collected sample (which may contain targetmaterial) is or can be disposed between reservoirs such that washsolution can pass through or over a solid matrix carrying the collectedsample. For example, the collected sample can be disposed between anabsorbent material able to absorb wash solution and an absorbentmaterial or other reservoir containing a wash solution. The saturationdifferential between these reservoirs provides for a directionaltransport of dye and wash solution across the collection pad surface.Preferably the wash solution is drawn through or over a matrix bearing acollected sample by capillary action. In embodiments where thecollection surface and dry absorbent material are in direct contact, thedry absorbent material should have at least enough capacity to absorbsufficient dye and wash solution to wash the sample collection surface.In other embodiments, rather than involving capillary action in anabsorbent material to draw wash solution through a sample-bearingmatrix, a wash utilizes user-applied pressure that pushes wash solutionthrough the sample-bearing matrix.

[0037] The term “matrix” refers to a solid material suitable forretaining dye/target material complexes. In the context of thisinvention, a matrix is preferably, but not necessarily a porous matrixor porous material, meaning that the matrix is penetrated by a largenumber of passages of sufficient size to accept the passage of fluidssuch as water, but are preferably not so large that the matrix isfree-draining. Such a porous matrix may be, for example, a network ofinterwoven fibers such as paper, cotton swab, nylon or polyester mesh,or felt. Thus, the absorbent materials utilized in this invention, forexample, for absorbing fluids to provide a flow through a samplecollection surface are porous matrices or materials.

[0038] In the context of entrapment of complexes of target material anddye and the removal of unbound dye, the term “wash” or “washing” refersto a fluid transport of sufficient unbound dye to enhance the detectionof complexes. It is understood that, in many cases, excess washing ofdyed materials can remove bound dye in addition to unbound dye.Therefore, when implemented the washing is not so extensive that removalof bound dye interferes with the detection of the presence of targetmaterial using detection of the presence of dye retained in or on asolid support or matrix.

[0039] In certain embodiments, the sample collection matrix is anabsorbent material, e.g., an absorbent pad, or the surface of anabsorbent pad. In certain embodiments, the sample collection matrixbinds the target material, in others the sample collection matrixentraps precipitated target material or the surface of the matrixretains dye/target material complexes.

[0040] In yet other embodiments, the device is arranged such that thesample collection matrix of the sampler is washed by wash solution bydiffusion, which may be assisted by physical agitation. Generally insuch embodiments, the sampler would then be removed from the dye-bearingwash solution. The target material, e.g., protein, would be bound to,entrapped by, or otherwise immobilized on or in a portion of, thesampler.

[0041] By “entrapment” or “entrap” is meant a physical association,which may be chemical, electrostatic or steric in nature, such that atarget material is retained in a matrix even in the presence of forcesthat otherwise might have a tendency to remove such target away from thematrix. This can occur, for example, through precipitation of targetmaterial such that the material becomes insoluble, e.g., usingprecipitating dyes such as Ponceau-S. In this way, washes may beperformed to separate small, unreacted or unbound dye molecules fromlarger, dye/target material complexes, thus facilitating testing ofsamples.

[0042] The term “precipitate” or “precipitation” as used in thespecification and claims includes the usual understanding ofprecipitation as a settling or deposition of solid particles out ofsolution. Additionally, the term as used herein also includes anygeneral retention of solid or particulate matter, by any force, within,or in some cases on, an absorbent collection pad matrix or sampler orother solid phase surface. Thus, the definition includes but is notlimited to target matter coming out of solution, target materialagglutination, and target material conformational changes that act toobstruct the exit of these materials out of a matrix by creatingcomplexes or other physical structures which cannot readily move throughthe pores of the porous material. Thus, those skilled in the art willreadily be able to select appropriate materials and conditions forprecipitation or other entrapment of a particular target material/dyecombination, e.g., selection of a porous material with an appropriateaverage pore size which will allow target material to penetrate into theporous material, but small enough to prevent dye/target materialcomplexes from quickly being transported out of the porous material orbinding of indicator dye to the matrix (e.g., absorbent pad or suitablecovering membrane).

[0043] In embodiments making use of a target material precipitating dye,e.g., protein precipitating dye, the dye stains/colors and immobilizestarget material, e.g., a protein (e.g., protein adsorbed to orprecipitated on an absorbent swab or pad). In this context,“immobilizes” means that the target material is removed from orprevented from entering the bulk of a solution (e.g., a dye solution orwash solution), such as by precipitation of the target material/dyecomplex, entrapment of the target material and/or target material/dyecomplex, or attachment of the target material to an insoluble or solidmaterial, e.g., a particle, matrix, or support. In embodiments wheretarget material binds to a matrix or surface, a precipitating dye neednot actually precipitate a target material as it is immobilized by thebinding to the solid matrix or support.

[0044] Certain of the embodiments described below demonstrate that thescope of the invention may also contemplate some minimum manipulation ofdevice components and devices in which the device does not remain sealedafter sample insertion and/or in which separate manipulation of one ormore device components is needed. Thus, the device in one embodimentincludes a sampler for collecting a target material or contaminant, asignal generator for providing a target material binding dye, a samplerwasher for washing unbound dye away from dye which is bound to targetmaterial and/or wetting agent reservoir, and at least one housing tocontain the signal generator and sampler washer and/or wetting reagents.

[0045] In certain embodiments, the sampler may take the form of a wand,wick, stick or any other configuration that is suitable for taking up aparticular type of sample. Such a sampler wand is generally an absorbentstick, preferably flattened, with a sample collection pad or surface ona terminal portion of the stick. The stick may also have an absorbentmaterial in communication with the sample collection pad/membrane, e.g.,on the other side of the same end of the stick, with communicationthrough a hole or holes in the stick. Thus, in these configurations,there is an absorbent pad or is material which is, or is adapted to be,juxtaposed to the collection surface for drawing target material and/ordye and/or wash solutions across the target material, e.g., a proteincontaminating a surface, and can facilitate entrapment of this targetmaterial on or within the pad matrix. The device incorporates a samplerwasher to wash unbound dye from the sampler collection pad or preferablya wetting agent reservoir, preferably into an absorbent pad orreservoir, which in some embodiments, such as the wand, is juxtaposed toor integral with the sampler collection pad/membrane, and in others isan elongated extension of the sampler collection pad or an abuttingabsorbent material housed by a sampler stick or housing and providingfor a flow of wash solution across the sample. Optionally, the devicefurther includes both a wetting agent or solution and sander washerwherein the wetting agent can be utilized to wet the sample collectionmatrix or surface. Such wetting can assist in sample collection and/orin picking up a quantity of dry dye. A wetting solution can be the sameor different from the wash solution. Thus, in applications where amoistened sample collection matrix is desired, the sample collectionsurface or matrix can be pre-moistened or can be moistened using awetting solution.

[0046] As indicated above, in particular embodiments, a sampler wand maybe constructed either with a sample collection pad/membrane but noadditional absorbent material, or with both a sample collection pad andan absorbent material for drawing fluids through the sample collectionpad, e.g., with a sample collection pad on one side in communicationwith an absorbent pad on the other side. Generally a sampler wand has ahandle, preferably made of a non-porous material such as variousplastics, coated papers, glass, or metal. Preferably the handle is atleast one inch long, and more preferably 2, 4, 6, or 8 inches long. Inother embodiments, such as ones including a sampler stick, there is nosandwich of the type described above. Rather, the body or housing of thesampler is hollow or integral with an internal reservoir adjacent orconnected to the sample collection surface for receiving or flushing thedyed sample of unbound dye with washing solution, or for providing awetting agent to the membrane.

[0047] Thus, the term “sampler stick” refers to an elongated housingstructure which includes a sample collection surface, pad or membrane,and at least one reservoir. For example, a sampler stick may contain awash solution, and/or a wetting agent, along with a sample collectionpad. The wetting agent or wash solution may be in continuouscommunication with the collection pad or may be separated with aseparator until communication is desired. Alternatively, a sampler stickcan contain a reservoir with dry absorbent material for absorbing washand/or wetting solution in communication with the sample collectionsurface. Exemplary sample sticks are shown in FIGS. 9-12.

[0048] In certain embodiments, the device includes a housing with aplurality of reservoirs, e.g., three reservoirs containing wettingagent, dye (dry or in solution), and wash solution. Alter taking up asample and a quantity of dye, the sample collection surface of thesampler is merely pressed against a wash solution reservoir to flushunbound dye from the sample, as the sampler itself unlike theembodiments above, possesses a complementary receiving reservoir. Thesampler stick formats, exhibited herein, are illustrative, but notlimiting. As an alternative, the reservoir in the sampler stick cancontain wetting/wash solution, and, following sample and dye uptake, thesample collection surface is pressed against an absorbent material in ahousing to cause a flow of wash solution across the sample.

[0049] Such sampler sticks can utilize a housing containing reservoirsin a planar arrangement, e.g., as shown in FIGS. 3 and 4, or can use ahousing in the form of a cap (e.g., FIG. 5). For example, such a cap canbe reversible, such that in one orientation the cap seals and/orprotects the sample collection surface. In the opposite orientation, thecap provides contact with dye and wash solution. Those skilled in theart will recognize that a variety of arrangements can be used to providemoistening of the sample collection surface, dye uptake or transportonto or into the sample collection surface and matrix, a source of washsolution, and a complementary absorbent material to receive washsolution as it washes the sample of unbound dye.

[0050] In some embodiments, certain of the reservoirs and/or reagentsare contiguous or adjacent but separated by rupturable membranes orseparators that, when broken, permit the flow of reagents across acollected/exposed sample to effectively wash the sample or wet thesample collection surface. FIG. 9 is exemplary but not limiting.

[0051] Some embodiments make use of solid dye which is hydrated andpresented to a sample in response to a physical stimulation such as arupturing of a membrane or membranes which maintain the dye in a dried,segregated state. FIG. 4 is illustrative, although by no means intendedto be limiting. (The combined dye/wash solution reservoir in FIG. 4could contain dry dye or a dye solution.) For example, a moistenedsample collection surface can be touched to a dry dye such that aquantity of dye is transferred to the sample collection surface. The dyecan contact target material directly and/or by fluid transport through aporous matrix to contact target material within the porous matrix.

[0052] The foregoing embodiments preferably utilize the properties ofprecipitating dyes but provide surprisingly good results with certainclasses of frequency shift dyes. As illustrated by those embodiments,the invention also provides methods of using such dyes to fix or retardthe egress of target materials, e.g., protein, from porous matrices intowhich target material has already been introduced, e.g. by swabbing.Thus, the introduction of target material as contemplated by the instantinvention is not facilitated by or dependent on movement of particles ormolecules in an electric field. Likewise, the method does not utilize aseparation of components of a sample due to differential migrationwithin the porous matrix. Instead, the matrix need merely be compatiblein size to allow the initial ingress or association of target withmatrix , and the influence of dye acts to thwart or inhibit the targetmaterial from leaving the matrix. This may be due, for example, toprecipitation, conformational changes, agglutination, or any otherresult of dye binding which has the effect of sufficiently immobilizingtarget material in the porous matrix that unbound dye can be washed awayand dyed target material visualized in the matrix. Alternatively, theimmobilization can be due to chemical or electrostatic binding of targetmaterial to matrix. Further, matrix constituency is irrelevant as longas the criteria described above are met and as long as the dye isotherwise compatible with, or can be made compatible with, the matrices,e.g with neutralizing agent.

[0053] Illustrative but not limiting of the possible materials that maybe used for the porous matrices are those discussed below under thedefinition of “sampler”. The dye should not bind to the matrix materialto such an extent that dye bound to target material in the matrix cannotbe distinguished from dye binding to matrix.

[0054] In accord with the aspects above, the immobilization orentrapment of target materials with a solid matrix, e.g., in a porousmatrix, provides a method for detecting the presence of target materialin a sample. As previously indicated, the method involves entrapping orotherwise immobilizing target material/precipitating dye or frequencyshift dye complexes on or within a solid matrix. For example, suchcomplexes can be entrapped in a porous matrix by binding of targetmaterial with precipitating dye or by collection of dye/target materialcomplexes on or in a collection surface or porous matrix. Generally,when using precipitating dyes the method includes washing away unbounddye to allow convenient visualization or other detection, e.g.,detection using an instrument such as a spectrophotometer orfluorometer. When using frequency shift dyes only wetting samplecollection and visualization is required. The method can involve variousmatrix materials, neutralizing agents, wash solutions and/or wettingsolutions, and dyes as described herein for other aspects.

[0055] For the methods herein involving precipitating dyes, azo dyes,preferably diazo dyes, which preferably have at least one, andpreferably a plurality of sulfonic acid groups, e.g., 2, 3, or 4 groups(which may be prepared in the corresponding salt form) are preferred.The red dye, Ponceau-S, Sigma Chemical Co., St. Louis, Mo. (chemicalabstracts service registry number 6226-79-5,(3-hydroxy-4-[2-sulfo-4-(4-sulfophenylazo)phenylazo]-2,7-naphthalenedisulfonic acid, tetrasodium salt],HOC₁₀H₄(N=NC₆H₃(SO₃Na)(N═NC₆H₄SO₃Na))(SO₃Na)₂, F.W. 760.58) isexemplary. Ponceau-S is soluble in water, slightly soluble in ethanol,and insoluble in vegetable oils. It is stable at room temperature inacetic acid and in preferred embodiments is used to stain proteinaceousmatter using a dye concentration of about 0.1-1.0% (w/v) in about 1-5%(w/v) acetic acid. The stain may be quickly removed upon addition of 0.1N NaOH, or by excess wash solution. The terms “azo dye” and “diazo dye”have the meanings as generally accepted in the dye industry. The term“sulfonated” in connection with the dye compounds refers to the presenceof sulfonic acid substituent groups. Such groups may be present in acorresponding salt form.

[0056] Advantageously, Ponceau-S binds rapidly to proteins andprecipitates or immobilizes them in addition to staining/coloring them.Thus, such a precipitating dye is generally used to bind to andprecipitate target material, e.g., protein, in or on a solid matrix. Insuch case, unbound dye is generally washed away from dye/proteincomplexes, providing visual detection of sample protein. In suchembodiments the dye does not bind to the solid matrix to such an extentor under such conditions as to prevent or interfere with detection ofdye/protein complex.

[0057] The invention provides a method for detecting protein on a solidsurface. Preferably the method is applied in testing for contaminationon to surface, e.g., food processing residue. The method involvescontacting a solid surface, e.g. a metal surface, with a Ponceau S dyesolution under conditions in which Ponceau S dye binds to protein. Therapid binding of Ponceau S to protein allows sufficient dye to bind toprotein even on vertical surfaces. Preferably the method allowsimmediate visualization of protein-bound dye on the surface withoutfurther processing. If desired, the method can further include washingthe surface with a wash solution which can wash away unbound dye.Preferably the dye is used at a concentration of 0.1-1.0% in diluteacetic acid. The dye solution and/or a wash solution can further containneutralizing agent as described above.

[0058] In certain other embodiments of the present invention, thebinding of dye to target material is detectable by a color change of thedye or dye solution, e.g., by a frequency shift of the dye on binding(color change) to target or of a dye solution by dye depletion.Preferably a self-contained sampling/testing device incorporates afrequency shift dye. Frequency shift dyes have their absorption orreflection or emission changed on interaction with target material,thereby differentiating bound from unbound dye. Such dyes can allowconvenient detection of target material even without separation of boundand unbound dye, thus not requiring a wash solution.

[0059] Therefore, in certain embodiments, the sample wash is able totransport sample material, e.g., target material from the collectionportion or surface of the sampler. Device embodiments whereinliquid-phase analysis is performed typically employ a reading portion ofthe device that permits the sample reaction to be visualized oranalyzed, with or without the aid of an instrument such as aspectrophotometer. The sample material is washed into the readingportion or alternatively carried into the reading portion on thesampler.

[0060] In certain other embodiments employing frequency shift dyes orother dyes where a change in dye color is to be detected, the samplermay be contained within a lower housing that provides protection for thesampler from pre-testing contamination.

[0061] Additionally, an upper housing may sealably engage the lowerhousing such that the two housings are in communication during the test.The sampler is preferably fixed to the upper housing. Within orcomprising such housings may be a chamber or reservoir to hold a wash ora wetting solution or a combined sample wetting signal generator orseparate reservoirs to hold each of a signal generator and a wettingsolution. A chamber may further include a breakable shaft contiguouswith the chamber that, upon breakage, exposes an orifice through whichthe contained solution may flow to the sampler and may further flow to aread portion for evaluation. Thus, the wetting solution can flow througha hollow shaft in a swab in the device, flow through the swab tip to thetarget collection pad/membrane/surface.

[0062] Frequency shift dyes provide convenient detection of bound dyeeven in the presence of unbound dye when the frequency shift is largeenough to distinguish the two. In cases where an instrument is to beused to read the binding results, the frequency shift can generally besmaller than if a visual reading is to be utilized. For machine reading,preferably an absorption shift on binding (expressed as a wavelengthshift) is at least 20 nm, more preferably at least 50 nm, still morepreferably at least 75 nm, and most preferably at least 100 nm. Forvisual reading, preferably an absorption frequency shift on binding isat least 50 nm, more preferably at least 75 nm, still more preferably atleast 100 nm, and most preferably at least 120 nm. For example, anabsorption peak of COOMASSIE blue stain under acidic conditions shiftsfrom about 465 mn to about 595 nm on binding of the dye to protein. Fora visual reading it is preferable if the absorbance change produces acolor change rather than just a shade change. For example, the GELCODE®reagent changes from amber to blue on protein binding. A fluorescentemission shift is preferably at least 20 nm, more preferably at least 40μm, still more preferably at least 75 μm, and most preferably at least100 nm.

[0063] GELCODE® includes colloidal COOMASSIE® G-250 dye. ColloidalCOOMASSIE® blue dyes may also be formed as described in the art. Forexample, in Neuhoff, et al., Electrophoresis 6:427-448 (1985) and inNeuhoff, et al., Electrophoresis 9:255-262 (1988). In general, thesesolutions utilize COOMASSIE blue dye® in an acidic aqueous solution withammonium sulfate or ammonium iron sulfate. In one example, the solutioncontains 0.1% weight/volume (w/v) COOMASSIE blue G-250 in 2% w/vphosphoric acid, and 6% w/v sulfate. In an alternative solution, the dyecontains 10% w/v ammonium sulfate and 20% w/v methanol. Preferably, thepH of the solution is between land 2 and the ammonium sulfate orammonium iron sulfate concentration is between 2% and 15% morepreferably between 4 and 10%, and most preferably between 5 and 8% w/v.The pH should not be so low that the dye molecules are rapidly degradedand the ammonium sulfate concentration should be selected so that thesolution takes on a color characteristic or the colloidal form,preferably the majority of the dye molecules are present as colloidalparticles rather than being in free solution or precipitating out ofsolution.

[0064] Another class of frequency shift dyes has been referred to as“protein error” dyes in the literature. The shift in the observedabsorption peak upon binding proteins is believed to be due to a shiftin pKa of an ionizable group or groups on the dye. This family includesbut is not limited to the triphenyl methane dyes, such as bromophenolblue, bromocresol green, and tetrabromophenol blue. In one embodimentutilizing this class of dyes, the present invention concerns aself-contained device having a sampler for collecting a sample which maycontain a target material, and a signal generator having a contactabledye that binds to the collected target material. In this embodiment thedye is bound either covalently or non covalently at or near the surfaceof the sampler (FIGS. 9-12). The dye could be either bound directly tothe absorbent material or bound to a membrane which covers all or partof the sampler surface as in FIGS. (9-11). The membrane should haveseveral characteristics in order to be useful in this application.Firstly, it should either bind the dye directly through non covalentforces or posses a structure which allows modification to acceptcovalent attachment of a dye. Secondly, the membrane should be somewhatporous, allowing passage of excess wetting agent and other solutesthrough to the absorbent material. Thirdly, since it is in contact withthe surface to be tested for protein, it must be resistant to excessivefraying. Examples of such materials include, but are not limited to,nylon, rayon, others polyesters, cellulose nitrate, and celluloseacetate.

[0065] For example, a nylon membrane is briefly exposed to a bufferedsolution of bromophenol blue at 5-500 μg/mL, preferably at 50-300 μg/mL,most preferably at 150-250 μg/mL. Although a preferred formulation ofthis buffer is 0.8 M NaCl, 0.1 M sodium citrate, 0.04 M acetic acid,4.3% (v/v) ethanol (pH 2.2), this is not limiting. An alternativeformulation of the buffer is 0.4 M NaCl, 0.1M citric acid, 4%ethanol (pH2.2). The membrane is then rinsed in a low pH buffer to retain itsyellow color and then it is dried and stored desiccated at roomtemperature. Under these coating and storage conditions the attachmentof the dye to the membrane is stable. The dye-containing membrane isthen placed at sampler collection surface and is in communication withor can be placed in communication with an absorbent material able totake up liquid from a wetting agent. The device may be constructed withany of many possible structural configurations, depending on therequirements of the particular application, e.g., depending on thespecific type of dye used and the type of target material to be tested.

[0066] The device may but need not also contain a wetting agent. In onesuch embodiment, the wetting agent is contained in a separatecompartment of the device (FIG. 9). Alternatively the wetting agent maybe stored in a separate housing of the device, access to which is gainedby puncturing or removing a seal (FIG. 10). The wetting solution may becontained in an ampoule which is contained in the housing. In addition,certain embodiments of the invention employ a pre moistened sampler suchas that depicted in FIG. 11. The wetting agent serves one or more ofseveral purposes including: (1) It may contain agents which facilitatethe release of the target material from the surface of the material tobe tested; (2) It may contain neutralizing agents which neutralize orpartially neutralize the deleterious effects of interfering materials;and (3) It will be of such a composition as to facilitate the frequencyshift of the dye in the presence of target material. In embodimentsusing these dyes the target (e.g., protein- containing) solution ispresented to the dye at a pH value just below that causing the colorchange. For bromophenol blue, the wetting solution should preferably bebuffered at pH 1.5-2.4, most preferably at pH 2.2 +/−0.2, and contain adetergent or detergents to solubilize the target, and a neutralizingagent or agents as described below. A preferred , but not limitingformulation of such a wetting solution is 0.1 M NaCl, 0.5 M citric acid,4.5 mM potassium sulfite, 3 mM sodium thiosulfate, 0.4% sodium dodecylsulfate, and 0.5% tergitol (pH 2.2).

[0067] As appreciated by one of ordinary skill in the art, certaindevice embodiments will accommodate the use of various types of targetmaterial binding dyes, e.g., precipitating dyes or frequency shift dyes.For example, embodiments which utilize a wash to carry sample materialaway from a sampler can be but need not be utilized with a frequencyshift dye. Such devices can also be used with a precipitating dye wherethere is the capability to wash unbound dye away from target materialbound dye in or on the sampler or on a porous separator. In accord withcertain embodiments described herein which incorporate a wash solutionin a reservoir in a sampler portion or upper housing, sample can becollected on a sampler, contacted with dye, e.g., from a reservoir inthe sampler portion or upper housing or by dye contained in the samplerprior to sample collection, and then washed by a wash solution containedin a reservoir in the sampler portion or upper housing. Preferably insuch embodiments, the collection surface of the sampler ispre-moistened.

[0068] Similarly, a device in which a dye solution is in a reservoircontactable with a sample collection surface (see e.g., FIG. 2 below)can be used with a frequency shift dye or with a precipitating dye. Witheither type dye, sample material is transferred to contact the dye onthe sampler. For a frequency shift dye, dye binding to target materialis detected by a color change of the dye as dye binds target material inthe dye solution or on the sampler, or as dye is depleted from thesolution as dye binds to target material in or on the sampler.

[0069] The terms “sampling/testing device” or “self-containedsampling/testing device” indicate that the device is constructed so thatall components for a particular assay are provided within a singledevice along with a means for introducing a sample into the device. Itmay, however, be advantageous for certain embodiments to utilize aseparate apparatus for incubation during the assay or for readingresults of the assay.

[0070] By “sampler” is meant a device component (or components) whichallows one to obtain all of or a portion of a sample which may bepresent on a surface, in a solution or in an atmosphere to be tested.For example, the sampler may be an absorbent pad, an absorbent pad whichhas a dye-containing membrane fixed to its surface, or a swab with ashaft and an absorbent tip. The shaft of the sampler or the samplerstick housing may be hollow, and may further include a vent. Asalternatives, the sampler/swab may take the form of a Q-Tip® or a simplepad. The swab may include natural or synthetic materials so long asdeposition of a sample there to may occur and dye binding to the swabdoes not interfere with detection of the target substance so as toprevent such detection. The absence or reduction of such interferencemay be provided, for example, by selection of material and/or by thephysical interrelationships of device components. The material may bebut is not limited to sponge, mylar, nylon, dacron, rayon, porex, porouspolypropylene, porous polyethylene, glass fibers, paper, or variousother woven or felted fibers such as nitrocellulose, cotton, wool,cellulose, or combinations thereof. In a preferred embodiment in whichthe swab includes a shaft, the swab shaft is preferably hollow, allowingthe sample wash and/or dye solution to flush the collected samplematerial from the swab into a reaction and/or read chamber or allowing awetting solution to moisten the sampler. The swab may be provided foruse in a pre-moistened form to assist in solubilizing and absorbingsample material into the sampler, or can be readily moistened from areservoir within the device containing a wetting solution, e.g., in asaturated absorbent matrix. The moistening fluid may be a buffer, water,acid, or base depending on the type of dye used. Those skilled in theart understand the selection of a compatible moistening fluid for thedye and target material involved in a particular type of test. Thesampler may function through capillary action, for example a capillarytube or tubes. The sampler may comprise a pipetting means. The samplermay comprise a chamber that captures a sample of an atmosphere, such asthe atmosphere present in an enclosed work space. The sampler may assumevirtually any shape or combination of shapes, e.g., planar, elongated,rectangular, circular, elliptical, cylindrical, spherical, cubical,conical, etc. Preferably the sampler is designed to enable a user toconveniently reach into locations in equipment, such as food processingor hospital equipment, which are difficult to access. Thus, the sampleris preferably constructed to provide an extension with a thincross-section, e.g., a cross-sectional area of less than 2 in², morepreferably less than 1 in², and in certain embodiments less than ½ in².Such extension is preferably least 2 inches in length, preferably least4, 6, or 8 inches in length. Such extension may be provided, forexample, by a wand handle, a swab shaft, or an elongated housing, orcombinations thereof.

[0071] The term “sampler portion” refers to a structural assembly whichincludes a sampler and also includes additional components which allowthe sampler to be sealed or attached to the remainder of the device, andmay also include one or more reagent spaces, such as a reservoir for asample wash solution.

[0072] By “sampler washer” is meant a device component (or components)which allows the removal of all or a part of a sample present on the“sampler”. For example, in some embodiments an upper housing or samplerportion or sampler stick comprises a chamber as a reservoir containing afluid in which the fluid may be selectively released as desired,ordinarily to release a sample that has been obtained or to wash unbounddye away from dye/target material complexes. In an alternativeembodiment, the upper housing or sampler portion may contain a containersuch as, but not limited to, an ampoule or a packet. The ampoule orpacket may contain a fluid as described above, which may be selectivelyreleased. In an alternative embodiment the upper housing or samplerportion may contain two containers, both or either comprising, forexample but not limited to, an ampoule or packet containing the same ordifferent fluids or dry substances. In another embodiment a fluid may bedirectly contained in the upper housing or sampler portion and acontainer or containers containing a fluid or dry substance (or morethan one fluid or dry substance) be contained therein. In yet otheralternatives, the lower portion of the housing or the lower housing maycontain a fluid that is used to wash the sample from the sampler, forexample, by inserting the end of the sampler into the fluid or otherwiseforcing the fluid against or through the sampler.

[0073] By “wash solution” is meant a solution, e.g., an aqueoussolution, capable of separating unbound dye from dye/target materialcomplexes and/or carrying sample materials from a sampler to anotherlocation. The solution may also serve the function of a wetting agent,e.g., for moistening a swab or collection surface in anticipation of orfacilitation of a sample collection. A wash solution for use with aparticular dye does not contain such amount of agents which tend todisrupt binding of that dye to target material that determination oftarget material binding is prevented. Preferably no such agents arepresent, but in some cases it may be desirable to include a low level ofsuch an agent or agents, for example, to minimize binding of dye to aporous matrix, thereby enhancing contrast and improving target materialdetection. Those skilled in the art will readily be able to select anappropriate wash solution for a particular dye. In embodiments utilizingPonceau S. dye, the wash solution and/or wetting agent are preferablydilute acetic acid solutions, preferably with 0.1 to 10% acetic acid inwater, more preferably 0.5 to 5%, still more preferably 1 to 5% aceticacid.

[0074] By “wetting agent” is meant an aqueous solution capable ofmoistening the sampler and/or hydrating dye to facilitate detection. Inthis context, the wetting agent does not remove unbound dye and thus isnot a wash solution.

[0075] By “signal generator” is meant a chemical compound or physicalstimulus or biological agent that provokes a measurable or discemableresponse in the presence of a target material; by chemical compound ismeant a chemical dye, an enzyme, or other organic or inorganic structurecapable of inducing such response.

[0076] The term “target material binding dye” or “dye” refers to acompound which will preferentially bind to a target material in a sampleas compared to binding to other molecules which are likely to be presentin such samples. Thus, the dye may bind to other molecules at a levelequal or greater than that for binding to the target material, but suchother molecules are ones which do not cause the characteristic change inproperty upon binding said molecule (e.g., frequency shift) and/or aregenerally not present in samples to be tested, such as samples to betested in evaluating process contamination. The preferential bindingneed not occur under all conditions, but at least occurs under the assayconditions selected for use in the device of the present invention. Thedye compound also is detectable using visual or spectroscopic means, andpreferably absorbs or fluoresces at visible wavelengths so as to give acharacteristic color. Binding of the dye to target material, e.g.,protein, preferably results in a color change and/or precipitation ofthe protein that is visible. Thus, for example, the term“protein-binding dye” refers to a compound that preferentially binds toprotein, polypeptides, or oligopeptides in preference to othermolecules. The dye, while active in aqueous form may be initially dryand hydrated during the testing process, e.g., when contacted withwetting/washing solution.

[0077] By “frequency shift dye” is meant a composition which uponinteraction with the substance to be detected exhibits a characteristic,detectable change in the light emission or absorption spectrum of thedye molecule. Preferably the alteration of the light absorptioncharacteristics of the dye molecule is observed. Changes in absorptionor emission spectra can include, for example, the appearance or increaseof absorbance or emission peaks or bands, the disappearance or reductionof absorbance peaks or bands and combinations of these. The frequencyshift dye may be a protein binding dye that is colloidal such asCoomassie® blue dye, preferably GelCode® Blue Stain Reagent PierceChemicals, Rockford, Ill., a “protein error dye” such as bromophenolblue, or other dyes whose spectra change in the presence of target.

[0078] The term “colloidal dye” refers to a dye which is in a finelydivided state in a liquid, such that the solid particles of dye are inthe range of 1 to 1000 nm, preferably in the range of 1-100 nm, and morepreferably in the range of 5-100 nm. This does not mean that all of thedye present in the liquid is in the form of such particles, as thoseskilled in the art recognize that the colloidal form is generally inthermodynamic equilibrium with solubilized dye and/or with solid dyeparticles larger than colloid size, e.g., larger than 1000 nm. Mostuseful are colloidal dyes where the amount of dye in colloidal form issufficient to alter the color of the dye solution as compared tosolutions containing the same amount of dye but in which the dye is insolution and/or in non-colloidal particles. In certain embodiments, atleast 30% of the dye molecules are in colloid size particles, preferablyat least 50%, and more preferably at least 70 % or 90%. As recognized bythose skilled in the art, a transition from soluble form to colloidalform of a molecule in liquid solution can be monitored by an increase inlight scattering of the solution.

[0079] In embodiments employing a frequency shift dye of the proteinerror dye family the wetting and or wash solutions are buffered at a pHnear that at which the color change occurs. For bromophenol blue, thewetting and/or wash solution (if present) are preferably buffered nearpH 2. At and below this pH the dye is yellow and with increasing pH thedye changes to blue/green. In the presence of protein the color changeoccurs at an anomalous pH. Thus, when bound to protein, bromophenol blueis blue/green rather than yellow at pH values near 2.0. A reasonableexplanation for this “protein error” is that binding of the dyedecreases the pKa of a group or groups whose ionization determines theabsorption spectrum of the dye, thereby causing the color change tooccur at an anomalously low pH value.

[0080] The term “swab” as used in the claims is used as a noun to denotean absorbent and/or adhesive pad that serves to collect sample targetmaterial in prelude to or concurrent with exposure to a signalgenerator, i.e., a dye.

[0081] By “neutralizing agent” is meant a chemical compound or solutionthat helps to neutralize potentially interfering compounds present onthe surface being tested or in a wetting agent present in or on asampler collection surface, which compounds may interfere with the dyebinding to the target material, e.g., protein. Exemplary neutralizingagents include sodium thiosulfate, sodium sulfite, sodium metabisulfite, Tween-20, sodium sulfate, sodium dodecylsulfate, tergitol(now called NiaProof Type 4), MgCl₂, and Triton-X® (Octoxynols;α-[4-(1.1 ,3,3,-Tetramethylbutyl)phenyl-ω-hydroxypoly(oxy-1,2-ethanediyl). All are available from Sigma, St. Louis, Mo. Triton-X®can be used preferably at an effective concentration of about 0.01-0.5%weight volume. Sodium thiosulfate may be used, preferably at aneffective concentration of about 0.01-1.0 mg/mL. MgCl₂ is preferablyused at a concentration of 0.2-20 mg/mL. These neutralizing agents maybe incorporated into any, all, or a combination of the wetting, wash,dye, or sample solutions provided. One of skill in the art willrecognize that other neutralizing agents may be substituted providedthey do not significantly interfere with the signal generator mechanismand measurement, and will understand what neutralizing agents areappropriate for a particular application or can determine whether or nota potential compound is appropriate by simple testing.

[0082] Thus, by “neutralize” is meant to inactivate potentiallyinterfering compounds present on the sample surface withoutsignificantly disrupting the signal generator's function in combinationwith the target material and the rest of the device.

[0083] By “effective concentration” is meant one that supplies, in wholeor in part, the intended or desired effect e.g., the desiredneutralizing effect.

[0084] The term “participates” as used in the claims denotes anassistance in the movement and/or gathering of target material onto thesampler, for instance by premoistening of an absorbent collection swabor pad.

[0085] By “reading portion” is meant a distinct section of the devicehousing wherein a reading or measurement or detection may be taken.

[0086] The term “in succession” connotes a temporal order but does notpreclude the use of wash solution as a wetting agent for a sampler swabin prelude to exposure to dye. Thus, the wash solution may be usedtwice, both before and after the dye.

[0087] The terms “contacted with”, “contacted by” or “on contact with”denotes the direct or indirect touching of one object with another.Certain embodiments have the contact mediated through a pierceablemembrane, which is rupturable by the sampler to effect the dyeing andwashing of a target material presented on the sampler.

[0088] The term “segregates” as used herein denotes a separation and/orcontainment which may be undone upon proper stimulation, for example thepiercing of a membrane by a sampler to allow mixing of components fromeach side of the membrane.

[0089] By “stably packaged” is meant that the dye or other signalgenerating component may be stored prior to use for prolonged periods oftime, for example, a year or more if stored at 4° C., and still providea signal upon activation. In one embodiment of the invention the signalgenerating means, e.g., comprising a colloidal dye solution, is stablypackaged within a sealed glass ampoule. The ampoule may be aborosilicate glass, for example Pyrex®. It may be an “onionskin” type ofglass ampoule. In other embodiments, a signal generating component,e.g., a dye, is sealed within a chamber by a membrane or membranes.

[0090] As understood by those skilled in the art, the stability of a dyemolecule will depend on the storage conditions, thus, the storage formcan be varied as appropriate for a particular dye. If the dye issufficiently stable in the test solution, the dye solution can bepackaged in the device as a single solution. Alternatively, if the dyeis not sufficiently stable in the assay solution, the stability can beenhanced by packaging the dye within the device separated from one ormore other components of the test solution until mixing is desired.Thus, for example, the dye and/or components decreasing dye stabilitycan be separated within the device by any of a variety of methods, suchas by using separate reservoirs or capsules or ampoules or separators orcombinations thereof, such that one or more can be ruptured, broken, oropened to allow mixing of various components at a desired time or times.

[0091] By “separator” is meant a device component(s) or structure forseparating two portions of the device, e.g., for separating the regioncontaining the sampler from the region in which detection is performeduntil introduction of the sample into the detection region (readportion) is desired or for separating a sample on the sampler from a dyesolution until contact between the sample and the dye is desired. Forexample, a separator may be a porous plastic or hydrophobic materialfilter, however, the porosity is not such that the sample would filterthrough without the application of a force, other than gravity, on thesample. As further examples, the separator may be a one-way valve, or apuncturable membrane or a breakable or rupturable reservoir or capsuleor ampoule.

[0092] A “reservoir” may be a well, ampoule, recess, void, or chambercapable of holding a liquid or solid. Such reservoir may be encased orcontained by a rigid, soft, or flexible housing such as a plastic. Areservoir may be or include an absorbent pad that is saturated orcapable of absorbing solution or solutions, e.g., target material, dye,wash, wetting agent, or combinations thereof. Such absorbent material ispreferably located in a depression, void, chamber, cavity or the like ofa housing.

[0093] Preferably the dye and test conditions are selected such that areadable result is provided within one hour at room temperature, morepreferably within 30 minutes or 20 minutes, still more preferably within10 minutes or 5 minutes, and most preferably within less than 1 minute.Such rapid results are particularly advantageous for field sanitationtesting, restaurant food preparation areas, hospital operating room,physicians' or veterinarians' offices and examining rooms, andlaboratory hygiene monitoring as retention of the samples for longperiods of time is not required and stability or consistency of the readof the completed test is enhanced.

[0094] As indicated above, a particularly advantageous embodiment of thepresent invention is adapted for protein detection, and thereforeprovides for the rapid and convenient testing of surfaces or solutionsfor contamination by protein-containing substances. The presence ofprotein can be a good indicator of residual food contamination remainingafter cleaning procedures have been completed, as protein is a componentof many food products. For example, in one application, the device willallow for testing of surfaces in food production plants, supermarketsand restaurants to ensure that cleanup procedures after food processinghave been effective. In other applications, the device would ensure thatappropriate cleaning of testing equipment or surfaces in hospital orveterinary operating rooms, examining rooms, or laboratory surfacesafter patient work-up or specimen testing was performed.

[0095] Certain embodiments utilize a dye capable of precipitating aswell as staining protein, for example, Ponceau-S. Ponceau-S isparticularly useful due to its speed of staining as well as its abilityto both precipitate and stain protein. Other embodiments utilizefrequency shift dyes such as bromophenol which rapidly change color inthe presence of target. Yet other embodiments are slower and utilizecolloidal protein binding dyes via a single step, integrated samplingassay device with visually distinct color changes in the presence ofsmall amounts of protein material, e.g. colloidal Coomassie® blue suchas found in GelCode® Blue Stain Reagent. Colloidal Coomassie Blueimparts a convenient spectral or color shift in the presence of protein.

[0096] By “protein” is meant peptide polymers (i.e., polymers of aminoacids) and thus includes oligopeptides, full-length cellularly-producedpolypeptides, degraded cellular polypeptides, complexes of polypeptides,and polypeptides associated with other molecules.

[0097] In certain embodiments, the results of a test using the devicecan be read visually. In other embodiments, the result can be read in aninstrument, such as a spectrophotometer, fluorimeter, or colorimeter.These devices are most useful for applications employing frequency shiftdyes.

[0098] The device in one embodiment includes a sampler and a combinedsample treatment, sample wash and signal generator stably packaged,preferably allowing easy visual interpretation.

[0099] By “a combined sample treatment, sample wash and signalgenerator” is meant components or structures to contain a targetmaterial binding dye, which preferably either precipitates and stainsproteins, or else creates a frequency shift on contacting protein, e.g.,colloidal dyes, protein error dyes. In either case, the dye is bound tothe sampler or the dye solution can be released at will to wash a sampleon or from the sampler thereby signaling the presence, absence, orquantity of protein present. In preferred embodiments the solutioncollects in a reading portion of the device. The dye solution or wettingagent can also treat the sample in a desired manner, for example, bysolubilizing or permeabilizing cell walls and/or membranes ofmicroorganisms (e.g., bacteria and fungi) or other cells.

[0100] By “fix” is meant that target material in the presence of aprecipitating dye, e.g Ponceau-S, is relatively slowed or halted fromdiffusing from or otherwise exiting the porous matrix in which it hasentered or is contained, as compared with target material/matrix in theabsence of dye.

[0101] Other specific applications of the invention include but are notlimited to the following: the testing of surfaces for other types ofcontamination such as carbohydrates, lipids and microorganisms; thetesting of liquid solutions for the presence of proteins, carbohydrates,lipids and microorganisms; the testing of air or gas for proteins,carbohydrates, lipids and microorganisms; and the testing of othermaterials such as dirt, vegetable material, manufactured articles,spices, powders, chemicals, debris and other types of samples familiarto those skilled in the art for such contaminants as protein,carbohydrates, lipids, nucleic acids, microorganisms, toxins, poisons,byproducts, adulterants and other materials recognized by those skilledin the art and capable of binding to dyes or ligands capable of beingcontained in colloidal or other forms which sequester or contain suchreagents so that reaction with specific target materials results in orcan be stimulated to result in rapid, detectable and distinct changes.

[0102] In accord with the provision of sampling/testing devices asdescribed above, in another aspect, the invention provides a method ofmaking such a test device by depositing a target material binding dye,preferably a precipitating-type dye or a frequency shift dye within areservoir in a self-contained sampling/testing device or upon or withinsurface to be contacted by the sample (e.g., membrane, pad, etc.).

[0103] As indicated above, for device embodiments which include asampler portion and a housing, the dye solution may be deposited in areservoir in the sampler portion or alternatively in the housing. Inembodiments in which the device includes an upper housing and a lowerhousing, the reservoir may be in the upper housing or alternatively inthe lower housing. The dye used is preferably a precipitating dye, suchas Ponceau-S. Alternatively, a frequency shift dye such as the proteinerror dye, bromphenol blue, or a colloidal dye such as colloidalCoomassie® blue is used which exhibit frequency and color shift changean contact with protein.

[0104] In another aspect, the invention provides a method for detectingthe presence of a substance, i.e., a target material, by using asampling/testing device as described above. Thus, in this embodiment,the method involves obtaining a sample which may contain the substanceto be detected (i.e., the target material) on or in the sampler.Depending on the type of sample, this may, for example, involve swabbinga surface, depositing a solubilizing or suspension liquid on a surfaceand then taking up at least a portion of that liquid, or taking up asample of liquid from a bulk liquid, such as by pipetting or in acapillary tube or by moistening an absorbent material. The sample iscontacted with a target material binding dye, e.g., a precipitating dyeor a frequency shift dye, as described above within the device, and thepresence of the target material in the sample is determined by detectingthe occurrence of a dye color change, e.g., a dye frequency shift, byobserving a visually detectable change in the color or shade of the dyesolution following contact with the sample or by reading a change orchanges in an absorbance or emission spectrum of the dye in a readinginstrument following contact with the sample or by detecting thepresence of bound dye on or in a matrix which immobilizes dye/targetmaterial complexes but which allows unbound dye to be separated, e.g.,by washing away.

[0105] In particular, a method of sanitation testing is provided inwhich a device as described above is used to detect the presence ofcontaminants on or from a surface or in solutions, such as followingcleaning procedures on a surface. In particular embodiments, the methodinvolves contaminant testing of surfaces or solutions in food processingfacilities such as a food production plants, restaurant, hospitals,laboratories, physicians' offices, ambulances, or veterinary hospitals.In preferred embodiments, the contaminant to be detected is proteinresidue.

[0106] The devices of the present invention generally are constructedsuch that a sample to be tested can be obtained on or in a sampler. Thedevice is constructed so that any remaining steps involved in detectingthe presence of a target material in the sample can be carried outfollowing placement of the sample-bearing sampler within the housingwithout further addition of assay components. This description generallydescribes embodiments which include a precipitating or frequency shiftdye, but applies also to the use of target material binding dyesgenerally.

[0107] Thus, the device components are arranged so that the sample, orat least a sufficient portion of the sample to allow detection of thepresence of target material, is contacted with a dye. The mixture ispresent in, or is transferred to, a portion of the device where theresults can be read, e.g., visually or in a spectrophotometer, afluorometer, or other reading instrument. Specific embodiments aredescribed below and in the figures with device elements arranged inparticular ways. However, it is clear that the invention also concernsdevices with elements selected and arranged in other ways to accomplishthe above process. Thus, for example, the dye solution can be locatedsuch that it is used as the wash solution to carry sample from thesampler to the reaction reading portion, the sample can be directlydelivered into the dye solution (e.g., by pipetting a liquid sample intothe dye solution or by inserting the sample-bearing portion of thesampler into the dye solution), or a wash solution can carry sample fromthe sampler into the dye solution. In view of the description herein,those skilled in the art will understand how the specifically describedembodiments can be altered to provide each of these and other formats.

[0108] Embodiments described in FIGS. 1-5 are particularly adapted foruse with precipitating dyes, embodiments described in FIGS. 6-8 areparticularly adapted for use with frequency shift dyes such as colloidalCoomassie blue., and embodiments described in FIGS. 9-12 areparticularly adapted for the use of frequency shift dyes of the “proteinerror” type, such as bromophenol blue.

[0109] Referring to FIG. 1: the device includes a plastic housing (3)with three wells. One well contains the target binding dye (5) and anabsorbent dye pad (4). The second well contains only an absorbent pad(6). The third well, separated from the other two by a hinge, containsan absorbent pad (1) and wash solution(s). The housing (3) is covered bya foil seal (9) that is removed prior to use. A separate sampler wand(1) incorporates a sample collection pad (2). The sample collection pad(2) is moistened by contacting it with the wash solution pad (7). Thesampler wand (I) is then used to swab a sample surface, e.g., a foodcontact sample surface, removing and absorbing food residue into thesample collection pad (2). The sampler wand (2) is placed into thedevice housing (3) and contacted with the dye pad (4) for a few secondsto transfer dye (5) to the sample collection pad (2). The sampler wand(1) is then placed on the absorbent pad (6) and the wash solution pad(7) pressed against the backside of the sample collection pad (2).Pressure is maintained for several seconds allowing wash to be drawnthrough the sample collection pad (2) into the absorbent pad (6),removing unbound dye. The sample collection pad (2) is ten observed forthe presence of color on its surface. The presence of colored dye isindicative of the presence of target binding material. In thisembodiment, the sample collection pad is selected such that targetmaterial will remain immobilized on and/or in the pad matrix during awashing step.

[0110] Referring to FIG. 2: The device includes a sampler wand (I) and aseries of three reservoirs in a housing/reagent tray (17). One reservoircontains wetting or washing agent/solution (14) used to moisten thesample wand before swabbing the surface. The second reservoir containsthe dye reagent (15). The third reservoir contains a wash agent/solutionthat may or may not be identical to the wetting/washing agent (16). Thereagents are localized in absorbent pads (18) at the bottom of theindividual well/reservoirs. The reagent tray (17) is covered by a foilseal (19) that is removed prior to use. The sample wand (10) comprises acollection surface (11) that abuts an absorbent pad (3) and the twopieces are held in place by a pad housing (12). The test is performed bymoistening the collection surface (11) of the sampler/wand (10) in thewetting/washing agent (14). The surface to be tested is then swabbed andthe sample on the collection surface (11) is then contacted with the dye(15) and then the excess dye is washed away when the surface issubsequently placed into the wash well (16). In each case the dye andwash agents are moved to/through the collection surface by absorptioninto absorbent padding or material (13).

[0111] Referring to FIG. 3: The reagent device is essentially the sameas in FIG. 2; however, the sampler (20) is in the form of a stickinstead of a wand, wherein the collection pad (23) surface may bedistinct from but contiguous with an absorbent material (22) encased bya stick housing (21). Alternatively, the absorbent collection pad (23)material extends down into the housing (21) and provides an absorptivedraw. Otherwise, this embodiment is manipulated in the same manner forwetting of the collection surface, treating the sample with the dye, andusing the wash reagent to wash away excess dye as in FIG. 2.

[0112] Referring to FIG. 4: The device uses the sampler stick as FIG. 3,but has the reagent housing (34) with 2 instead of 3 wells. The firstwell contains the wetting reagent (35) that is used for moistening thecollection pad (33) surface, and the second well has two compartmentsvertically arranged, with the dye (36) layered on top of the wash (37).The dye is present in dry form on top of a breakable membrane (36 a).Following contact with the dye, the membrane (36 a) is pierced with thesampler stick and wash solution (37) is absorbed into and through thecollection pad (33).

[0113] Referring to FIG. 5: This device is an embodiment that has thereagents in the reagent tray/housing (46) that is in the shape andfunction of a cap, as well as inside the sampler stick (40). The reagenttray/housing (46) in this embodiment fits onto the end of the samplerstick (40) as a reversible cap. The collection pad surface (45) ispre-moistened with wetting agent (51). In the exemplary form, thecollection pad surface (45) is covered by the end of the reagent tubehousing (46) with a breakable membrane (47) protecting the collectionsurface (45) from the dye (48) in an absorbent pad (49). The samplestick (40) is removed from the reagent tube housing (46) and used tocollect the sample. The reagent tube housing (46) is then put back onthe sampler stick (40) after being rotated 180 degrees and the same sideof the cap is placed on the sampler stick employing the alignment guide(42). The collection pad surface (46) pierces an initial barrier (47),thereby coming into contact with dye (48) and taking a quantity of thatdye on the collection surface (45). Then the sampler stick (40) is putonto the other end of the cap at which point the breakable seal (43) inthe sampler stick housing (41)is broken, allowing the wash reagent (44)in the sampler stick (40) to migrate through the collection pad (45)surface and into the absorbent material (50) in the cap/reagent trayhousing (46).

[0114] This effectively washes away excess dye, so that only the dyeremaining of the collection surface is dye which has been immobilizeddue to binding to target material, e.g., protein.

[0115] Referring to FIG. 6: This embodiment of the device (60) includesa sampler portion or upper housing (61) a dye reservoir (62) containingthe target binding dye (70); an orifice (64) communicating with thehollow swab shaft (66), exposed by breaking off the snap plug (68); ahousing (74); an absorbent swab tip (72); and a lower read chamber orread portion (76).

[0116] Referring to FIG. 7: In another embodiment the device includes anupper housing (80), an upper barrier means (81) between the upperhousing (80) and the upper section (82) of a lower housing (87). Theupper housing (80) and upper barrier means (81) define a chamber (88). Asampler (83) is attached to the upper housing. The lower portion of thelower housing (87) forms a read portion (85).

[0117] Referring to FIG. 8: This embodiment is as in FIG. 6, except thatthe dye reservoir (62), contains a wash solution (71) which does notcontain a dye. The housing contains a foil barrier (78) (i.e., aseparator) dividing the housing into an upper section (73) and a lowersection (75). The lower section contains a dry dye (79), and forms asealing, slidable junction with the upper section (73). In thisembodiment, other types of barriers can be used to prevent the washsolution from washing the sampler before such washing is desired.Similarly, other types of separators can be used to divide the housinginto upper and lower sections. Also, the dye in the lower section can bea dye solution or suspension rather than a dry dye. The slidablejunction between the upper and lower sections of the housing may includea threaded surface(s) such that the upper and lower sections may bescrewed together, thereby piercing the separator with the sampler.

[0118] Referring to FIG. 9: This embodiment is as in FIG. 4, except thatthe dye is bound, either covalently or non covalently to a membrane (89)which is in communication with the absorbent material (90) on thecollection surface of the sampler (91). The membrane and absorbentmaterial are inserted into a plastic housing (92). Together thesecomponents form the sampling stick (93). In this embodiment a reagenttray housing (94) is employed. This tray contains an absorbent pad (95)containing wetting agent (96).

[0119] Referring to FIG. 10: This embodiment is as in FIG. 9 in that thedye-membrane (97) is in communication with the absorbent material (98)on the collection surface (99) of the sampler tube (100). In thisembodiment the wetting solution (101) is contained within a sealedcompartment in the plastic sample housing (102) and is separated fromthe absorbent material and thus the dye-membrane by a breakable seal(103).

[0120] Referring to FIG. 11: This embodiment is as in FIG. 10 in thatthe dye-membrane (104) is in communication with the absorbent material(105) on the collection surface (106) of the sampler tube (107). In thisembodiment the wetting solution (108) is contained within a breakableampoule (109) within a sealed compartment in the plastic sample housing(110).

[0121] Referring to FIG. 12: This embodiment is as in FIG. 9-11 in thatthe dye-membrane (111) is in communication with the absorbent material(112) on the collection surface (113) of the sampler tube (114). In thisembodiment the wetting solution (115) is used to pre-moisten theabsorbent material (112) and thus the dye-membrane (111). To preventevaporation of the wetting solution (115), the sampler tube or samplertubes are sealed within an air-tight pouch (116).

[0122] In addition to the embodiments described in the figures,additional embodiments can be constructed with various combinations andarrangements of elements which also accomplish contacting a sample witha target material binding dye, e.g., a precipitating or frequency shiftdye, within the self-contained sampling testing device. Exemplaryselections and arrangements are described. In accord with theembodiments described above, a device may be constricted to include asampler portion which sealably attaches to a housing, or may beconstructed as an upper and a lower housing in which the sampler isattached to the upper housing and the upper and lower housing sealablyengage. Other variants can also be constructed.

[0123] As previously indicated, in the various embodiments differenttypes of samplers can be utilized. These include, for example, swabs,pipettes and capillaries. For embodiments in which the sampler is a swabor other wiping device, a sample washer is provided. In certainembodiments, the sample washer includes a reservoir containing a washsolution that can be used to wash the sample from the sampler. Deliveryof the wash solution to the sampler can be accomplished in a variety ofways including, for example, rupture of a membrane to allow washsolution top through a hollow sampler shaft, or breaking the tip or plugto expose an orifice communicating with a hollow sampler shaft allowingthe fluid to flow down the shaft, or rupture of a packet or ampoulethereby releasing a fluid that can then flow down a sampler shaft towash the sample.

[0124] The wash or wetting solutions may also be constituted andpackaged in a variety of different ways as appropriate for variousconfigurations and dye selections. For example, as described above, thewash solution may include the dye. However, in certain embodiments itmay be preferable to package the dye separately from the wash solution.For example, the dye and other wash solution components may be separatedin the upper reservoir until mixing is desired. As an example, aconcentrated dye solution may be provided in a breakable ampoule orrupturable packet within a reservoir chamber containing other washsolution components.

[0125] Alternatively the dye and other wash solution components may bein separate chambers separated by a separator. Breakage of the dyecontainer or combining the contents of separate chambers, then resultsin mixing and thus provides a combined dye wash solution. Such anarrangement may be desirable, for example, where the dye molecules wouldnot have long-term stability in the presence of one or more other washsolution components. Alternatively, the wash solution and dye may beseparated by providing the wash solution only in the upper reservoir andproviding the dye in a reservoir or ampoule or packet or chamber in alower portion of the device, e.g, in a lower portion of the housing orlower housing.

[0126] In embodiments where the sampler is a pipette or a capillary thesample can be removed from the sampler in a variety of ways, such as byexpelling the liquid sample with air, or by washing the sample from thepipette or capillary with a wash solution. In general, to remove thesample, the upper portion of the device will be deformable to allow acreation of pressure to push the liquid sample from the pipette orcapillary.

[0127] Similar to the embodiment described above in which the dye isseparated from other solution components in the sampler portion of upperhousing, in embodiments where the dye is contained in a lower portion ofthe device the dye can be separated from other solution components byplacing either or both of the dye or other components into separatechambers, ampoules, packets, or other structures such that thecomponents can be mixed at a desired time.

[0128] In yet another embodiment, the sampler is directly inserted intoa solution in a lower portion of the device. For example, in certainembodiments the upper portion of the device does not contain a reservoirwith a wash solution. Instead, the wash solution with or without dye iscontained in a lower portion of the device and the sampler is insertedinto the wash solution following sample collection. In such embodimentsthe wash solution can be separated from upper portions of the device bya barrier, for example, a rupturable membrane or one-way valve ordeformable constriction through which the sampler can be inserted. Also,in such embodiments, as noted, the dye may be packaged separately fromthe wash solution or may be incorporated in the wash solution. Asdescribed before, such separation may be accomplished by the use ofseparate chambers, rupturable packets, breakable ampoules, rupturablemembranes, semi-porous filters, and other such structures.

[0129] The method of using one embodiment of the device to test for thepresence of protein will be briefly described. This embodiment of thedevice has the structure of the device illustrated in FIG. 9, andutilizes a dye bound to a membrane at the surface of the sampler todetect the presence of protein on a surface.

[0130] A sampler stick (93), stored in a plastic container or sealablepouch with desiccant is removed and the top of the reagent tray housing(94) is removed exposing the absorbent pad (95) containing wetting agent(96). The collection surface (91)/dye membrane (89) is briefly pressedagainst the absorbent pad (95) activating the device. An area (2 in ×2in) to be tested for protein is swabbed with a moistened collectionsurface (91), allowing a portion of the protein material bind to the dyeon the dye-membrane (89). The sampler (61) is then sealably engaged ontothe housing (74). If protein is present in the sample the dye on thedye-membrane (89) will change from a yellow color to blue within 1minute. If no protein is present the color of the dye-membrane (89) willremain yellow.

[0131] The device embodiments described herein are constructed from anyof a variety of materials or material combinations, including but notlimited to plastics. Injection mold castings or any other means forgenerating suitable device housings may be employed. In appropriatedevices, well/reservoirs may be machine-drilled or injection molded orformed by other methods suitable for forming such cavities in theparticular materials. Those skilled in the art are familiar and canselect suitable materials and construction techniques. Also whereappropriate, as in embodiments such as the book of FIG. 1, separatehousings and pieces may be joined by hinges, snaps, latches, Velcro®, orany other connector that does not impede the ability of the reagents tofunction. The absorbent swabs and collection surface materials, alreadydescribed, are comprised of any of the following illustrative materialsor functional equivalents thereof: sponge, mylar, nylon, dacron, rayon,porex, porous polypropylene, porous polyethylene, glass fibers, paper,or various other woven or felted fibers such as nitrocellulose, cotton,wool, cellulose, or combinations thereof. These may in turn be attachedto housings where appropriate, such as in the embodiments of FIGS. 1, 2,or 3, by glue, adhesive, or any other means which does not interferewith target material collection, staining or, in the case ofprecipitating dye use, the precipitation or other immobilization oftarget material.

[0132] Those skilled in the art will recognize that this and otherembodiments of the present invention can be used in a variety of ways,including the following:

[0133] (1) Testing of liquid samples to determine if they containcontaminating material. The procedure utilized to test for material in aliquid sample would be similar to the procedure used to test a surface,with the difference being that the sample tested is a liquid.

[0134] (2) Testing of any sample for contamination and using aninstrument read instead of a visual read.

EXAMPLES

[0135] Although the following examples are patterned after applicationsto the food industry, similar results would be obtained regardless ofthe source of protein (e.g., human or animal sera or other secretions,etc.

Example 1

[0136] Exemplary devices were constructed as generally described in FIG.2 and used with a precipitating dye (Ponceau 5) and a frequency shiftdye (a colloidal Coomassie Blue dye, Gelcode®) and used to test foodsurfaces soiled with milk, cheese, roast beef, turkey, or tomato. Thesurfaces were also tested with a food industry-accepted means ofmeasuring surface contamination based on ATP detection (LIGHTNING,produced by IDEXX Laboratories, Westbrook, Me.)(used according tomanufacturer's instructions) as well as the protein detection devicesdescribed for this invention. As indicated, two different embodiments ofthe present invention were used. One with Ponceau-S as theprotein-binding dye, and one with Gelcode®-a colloidal Coomassie bluedye.

[0137] Stainless steel surfaces were smeared with the indicated foodmaterials. For each test, a sample was obtained from the surface byswabbing with the moistened sampler collection surface of a sampler fromthe particular device. “Dirty” indicates that the surface was testedfollowing application of the food residue, to the surface; “wiped clean”indicates that the surface was wiped free of visible food residue with adry paper towel; and “scrubbed clean” indicates that the surface was wetcleaned with a brush and detergent type cleaning solution in a mannercommonly used for cleaning in the food processing industry.

[0138] For the Ponceau S device, the absorbent pad of the sampler wasmoistened with the wetting agent, a sample was swabbed from the surface,then the absorbent pad of the sampler was touched briefly (a fewseconds) against the dye. The absorbent pad of the sampler was thendipped in the wash solution to wash away unbound dye.

[0139] The Gelcode® device was used similarly except that the colorchange of the dye was observable both in the dye solution and on thesampler pad.

[0140] The results are shown in Table 1. The data indicate tat to deviceis able to distinguish the three different states of the surfaces(dirty, wiped clean, and the more 5 thorough, scrubbed clean) for eachfood type. Both dyes gave results that allow the test operator todistinguish between dirty, minimally cleaned (wiped) and thoroughlycleaned (scrubbed) surfaces. Results for the LIGHTNING® device rangefrom 0-7.5. Dye results are read by eye and assigned a numeric valuefrom 0-5. In both cases the higher the number, the greater the indicatedlevel of contamination. TABLE 1 Comparison of Bioluminescence assay(Lightning) to protein detection devices. Lightning results in zones(0-7.5). Dye results are read by eye and assigned a numeric value from0-5. Milk Cheese Roast Beef Turkey Tomato Biolumines- cence Lightningdirty 3.3 2.55 4.9 5 6.05 wiped clean 2.4 2.1 3.45 3 4.85 scrubbed clean2.05 1.65 1.65 2.05 2.35 Protein Detection Devices Ponceau S 3.5 4.5 4 31.5 dirty wiped clean 125 1.25 0.75 0.25 0.25 scrubbed clean 0 0 0 0 0Gelcode 4.5 4 5 4 3 dirty wiped clean 3.5 2.5 225 1.5 0.75 scrubbedclean 025 0 0 025 0.5

Example 2

[0141] An exemplary device constructed as generally described in FIG. 6and containing 2 ml Pierce Gelcode® dye was used in a test to determinedetection sensitivity of the device. Presence of protein was detectedusing qualitative visual reading and by reading the optical density (OD)at 595 nm, with the reported OD being the mean of two readings.

[0142] Bovine serum albumin (BSA) at various concentrations was dried onclean 4″×4′ stainless steel coupons. For each sample tested, thepre-moistened swab portion of a device was swiped over the couponsurface with firm pressure to collect the sample. The swab was insertedinto the housing, and the dye reservoir bulb snapped to the side todeliver the dye into the lower read chamber. A visual interpretation isthen made, followed by transfer from the read chamber to a disposablecuvette for reading at 595 nm. The results are shown in Table 2. SampleO.D. Visual Interpretation Negative control (PBS) 0.0006 Negative BSA, 5μg/test 3.3800 + + + + BSA, 50 μg/test 1.3570 + + + BSA, 10 μg/test0.4130 + + BSA, 5 μg/test 0.1930 + BSA, 2.5 μg/test 0.0900 +

[0143] BSA refers to Bovine Serum Albumin.

[0144] The results demonstrate that this embodiment of the device has adetection sensitivity of about 2.5 μg protein/test.

Example 3

[0145] An exemplary device as in Example 2 was used in a comparison testof biological contamination with the Konica Hygiene Monitoring Kit. TheKonica kit was utilized according to manufacturer's instructions withreading after 10 minutes at room temperature. The exemplary device wasutilized as follows.

[0146] Various different sources of protein were dried upon clean4˜×4˜stainless steel coupons, which had been marked to divide eachcoupon into two equal parts. The exemplary device was used to collectthe sample from the left side of the coupon surface. Following theKonica kit procedure, the corresponding right side of the coupon wassampled with the Konica swab. Visual interpretation for the exemplarydevice was made immediately upon activation. The Konica test was read at10 minutes according to kit instructions. The stainless steel couponswere then washed with a mild detergent (Palmolive®) and water, and afterdrying, each side of the coupon was retested to detect any remainingcontamination on the surface.

[0147] The results of the comparison test are shown in Table 3.Cleanliness levels for the Konica kit are shown according to acleanliness standard where:

[0148] Level I (Clean)

[0149] Level 2 (Less Clean)

[0150] Level 3 (Slightly Dirty)

[0151] Level 4 (Dirty) TABLE 3 Exemplary Sample Konica Kit Device Milkon coupon Level 3-3.5 (slightly + + + + dirty) 1 wash Level 1-1.5(clean) + + + 2 washes Level 1 (clean) Negative 5 μg/test BSA standardLevel 1 (clean) ± 1 wash Level I (clean) Negative 1:400 Plasma Level 1(clean) + + 1 wash Level 1 (clean) Negative

[0152] The results indicate that the device is more sensitive than theKonica test system, in addition to the advantages of being faster andmore convenient to we.

Example 4

[0153] An exemplary device as described in FIG. 9 was used in acomparison test of biological contamination with the Konica HygieneMonitoring Kit (AssureSwab).

[0154] The exemplary device was prepared as described in the text usinga nylon membrane which had been treated with a 200 ug/mL solution ofdye. The sampler surface was briefly exposed to wetting solution and thetest area (4 in×4 in) immediately swabbed. A transition from a yellowcolor to blue-green occurred within one minute if protein were present.The intensity of the blue-green color was assessed on an arbitrary scalefrom 0 (yellow) to 2 (deep blue-green).

[0155] The Konica kit was utilized according to manufacturer'sinstructions with reading after 10 minutes at room temperature.

[0156] Various different sources of the test protein BSA (bovine serumalbumin) were dried upon clean 4 in ×4 in sections of a plastic plate.The exemplary device was used to collect the sample from one section ofthe surface. Following the Konica kit procedure, the section was sampledwith the Konica swab. Visual interpretation for the exemplary device wasmade immediately upon activation. The Konica test was read at 10 minutesaccording to kit instructions.

[0157] The results of the comparison test are shown in Table 4.Cleanliness levels for the Konica kit are shown according to acleanliness standard where:

[0158] Level I (Clean)

[0159] Level 2 (Less Clean)

[0160] Level 3 (Slightly Dirty)

[0161] Level 4 (Dirty) TABLE 4 Sample Konica Kit Exemplary Device  10 μgBSA Level 1 (clean) 0.1  25 μg BSA Level 1 (clean) 0.3  50 μg BSA Level1+ (clean) 0.3 100 μg/BSA Level 2 (less clean) 1.0

[0162] The results indicate that the device is more sensitive than theKonica test system, in addition to the advantages of being faster andmore convenient to we.

[0163] All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

[0164] One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Thesolutions, dyes, and methods described herein as presentlyrepresentative of preferred embodiments, arc exemplary, and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in The art which areencompassed within the spirit of the invention as defined by the scopeof the claims.

[0165] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention. For example, those skilled in the art will recognize that theinvention may be practiced using a variety of different dyes, and pHbuffers, as well as additional reaction components.

[0166] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

[0167] In addition, where features or aspects of the invention aredescribed in terms of Markush groups or other grouping of alternatives,those skilled in the art will recognize that the invention is alsothereby described in terms of any individual member or subgroup ofmembers of the Markush group or other group.

[0168] Thus, additional embodiments are within the scope of theinvention and within the following claims.

What is claimed is:
 1. A self-contained sampling/testing devicecomprising: a. a sampler for collecting target material; b. a signalgenerator comprising a dye which binds to said target material to signalthe presence of said target material.
 2. The device of claim 1, furthercomprising a sampler washer comprising a wash solution.
 3. The device ofclaim 1, further comprising an absorbent material, wherein said samplercomprises a porous sample collection pad, and said absorbent material,said sampler and said sampler washer are configured and arranged suchthat said sample collection pad can be disposed between said absorbentmaterial and said sampler washer so that said wash solution separatesdye bound to said target material immobilized on said sample collectionpad from unbound dye.
 4. The device of claim 1 wherein said samplercomprises a porous sample collection pad for collecting said targetmaterial, wherein said dye and said wash solution are contained in areagent tray housing capable of being contacted by said sampler toimpart said dye and said wash solution across said target materialcontained on said sampler.
 5. The device of claim 3 or 4 wherein saiddye and said wash solution are contained in a plurality of reservoirs,wherein said reservoirs are serially contacted by said sampler to firstexpose said target material to said dye and then to wash unbound dyeaway from bound dye.
 6. The device of claim 1, 3, or 4, furthercomprising a wetting agent for moistening said sample collection pad inadvance of collecting said target material.
 7. The device of claim 6wherein said wetting agent is identical to said wash solution.
 8. Thedevice of claim 3, wherein said sampler is hollow and wherein saidabsorbent material is disposed within said sampler to facilitatetransport of said dye and said wash solution across said target materialor said wash solution is disposed within said sampler.
 9. The device ofclaim 8, further comprising a reagent housing comprising an absorbentmaterial, wherein one of said absorbent materials is saturated with saidwash solution, and the other of said absorbent materials is unsaturated,wherein unbound dye from a sampler collection surface disposed therebetween is washed by the flow of said wash solution from said saturatedabsorbent material to unsaturated absorbent material.
 10. The device ofclaim 9, wherein said dye is transported by said wash solution to saidtarget material to effect said binding.
 11. The device of claim 10,wherein said dye is separated from a said absorbent material by at leastone membrane that is rupturable during the course of a test.
 12. Thedevice of claim 9, wherein said reagent housing also comprises a cap forprotecting said collection pad surface from contamination when not inuse.
 13. The device of claim 3 or 4, wherein said dye is a proteinbinding dye.
 14. The device of claim 13, wherein said dye is Ponceau-S.15. The device of claim 14, wherein Ponceau-S is used at a finalconcentration of about 0.1-1% (w/v) in dilute acetic acid solution. 16.The device of claim 13, wherein said dye both precipitates and stainssaid protein.
 17. The device of claim 1, 3, or 4, wherein said dye isdry until contacted by said sampler.
 18. The device of claim 1, furthercomprising a neutralizing agent to neutralize any compounds in thesample that might interfere with the binding of said dye to said targetmaterial.
 19. The device of claim 18, wherein said neutralizing agent isselected from the group consisting of sodium thiosulfate, MgCl₂, sodiumdodecyl sulfate, tergitol, Triton X-100, and Tween
 20. 20. The device ofclaim 17, wherein said dye is a frequency shift dye.
 21. The device ofclaim 20, wherein said dye is a colloidal dye.
 22. The device of claim21, wherein said dye is a colloidal Coomassie® Brilliant Blue dye. 23.The device of claim 1 further comprising a reading portion.
 24. Thedevice of claim 22, wherein said sampler is contained within a lowerhousing providing protection from pre-testing contamination for saidsampler, said device further comprising an upper housing, wherein saidupper housing and said lower housing sealably engage, and said sampleris attached to said upper housing.
 25. The device of claim 1, whereinsaid sampler further comprises a hollow shaft and an absorbent tip; achamber housing said combined sample wash signal generator, said chamberfurther comprising a breakable shaft which upon breakage exposes anorifice through which said combined sample wash signal generator mayflow, wherein said chamber is adjoined to a slidably engageable fitting,said fitting having an inner member and an outer member between which alower housing may slidably engage, wherein said combined sample washsignal generator comprises a frequency shift dye; a lower housing readportion below said sampler to contain the combined sample wash signalgenerator, wherein said read portion comprises walls allowing detectionof a frequency shift of said dye.
 26. The device of claim 1, whereinsaid device comprises a surface which binds said target material priorto contact of said target material with said dye.
 27. The device ofclaim 26, wherein said target material is protein.
 28. The device ofclaim 1, wherein said sampler contains an absorbent pad at the surfaceof which is positioned a membrane to which a dye is attached eithercovalently or non covalently.
 29. The device of claim 1, wherein saidsampler contains an absorbent pad which has a dye attached directly toits surface either covalently or non covalently.
 30. The device of claim28 or 29, wherein a wetting/neutralizing solution is contained within areagent housing containing an absorbent material.
 31. The device ofclaim 28 or 29, wherein said sampler contains an absorbent materialwhich is pre moistened with wetting/neutralizing solution.
 32. Thedevice of claim 28 or 29, wherein said sampler contains a breakable vialor rupturable compartment containing wetting/neutralizing solution. 33.The device of claims 28-32 wherein said dye is a frequency shift dye ofthe protein error family.
 34. The device of claims 28-32, wherein saiddye is bromophenol blue.
 35. The device of claims 28-32, wherein saidwetting/neutralizing solution is selected from the group consisting ofsodium thiosulfate, MgCl₂, sodium dodecyl sulfate, tergitol, Triton X-100, and Tween
 20. 36. A self-contained sampling/testing devicecomprising: (a) a sampler for collecting biological target material; and(b) a signal generator comprising a frequency shift dye of the proteinerror family, said dye being capable of binding to said biologicaltarget material and producing a color change upon binding to said targetmaterial wherein unbound frequency shift dye need not be separated frombiological target bound frequency shift dye in order to detect thepresence of target material.
 37. The device of claim 36, furthercomprising an absorbent material.
 38. The device of claim 36, furthercomprising a wetting agent.
 39. The device of claim 36, wherein said dyeis bromophenol blue.
 40. The device of claim 36, further comprising awetting solution, said solution comprising a neutralizing agent selectedfrom the group consisting of sodium thiosulfate, MgCl₂, sodium dodecylsulfate, tergitol, Triton X-100, and Tween 20.